Altered expression of cathepsins B, and L is associated with breast cancer and many other human malignancies. These alterations involve changes in both transcription and intracellular trafficking such that increased amounts of these proteins are targeted to the cell surface or secreted into the extracellular space. The extracellular enzymes are thought to facilitate tumor growth or metastasis, perhaps through modification of cell surface or extracellular matrix proteins. While the association of cathepsins with cancer has been known for some time, little is known about how these proteins participate in cancer-related processes. The goals of the proposed research are to provide direct evidence for the involvement of cathepsins in breast cancer, to define the nature of the involvement, and to begin to explore the basis for cathepsin function at the cellular level. The proposed studies will utilize a unique set of mouse breast tumor cell lines which differ in their ability to progress through various stages of metastasis. In preliminary studies, the most highly metastatic lines of the set were found to secrete either elevated levels of metalloproteinases and plasminogen activators, or lysosomal cathepsins. Multiple phenotypes were observed for cathepsin secretion, indicating that multiple mechanisms are involved. For example, one of the cell lines (66c14) displayed generalized secretion of cathepsins and other lysosomal proteins, while another line (4T07) secreted cathepsin L selectively. To establish the involvement of cathepsins B, D and L in tumor progression, cathepsins will be overexpressed or ablated in these cell lines and the effects on in vivo growth and metastasis will be determined. Cathepsin overexpression will be accomplished by transfection of mouse breast tumor cell lines with cathepsin cDNAs, and cathepsin ablation will be achieved by gene targeting methodology. Molecular and cell biologic techniques will be used to determine the molecular basis for the observed alterations in cathepsin synthesis and trafficking and to further investigate at the cellular level how cathepsins participate in cancer-related progresses. From these studies, they hope to acquire (i) a better understanding of how lysosomal proteinases participate in the progression of cancer, (ii) an understanding of the cellular and molecular mechanisms responsible for cancer-related alterations in cathepsin expression and trafficking and (iii) information that may be of use for more effective diagnosis and treatment of the disease.